Thrombin is a crucial regulating enzyme in the coagulation cascade and plays a critical role in cellular activation and chemotaxis, as well as being involved in inflammation, tissue repair and neural cell viability. Thrombin's effects on cells are mediated by the thrombin receptor, which evokes second messenger signaling through G-protein coupling and protein phosphorylation. To determine the biological functions of thrombin and the thrombin receptor in vivo, gene targeting will be used to create genetic deficiencies of these proteins in embryonic stem (ES) cells and mice. These models will be used to study the biological roles of thrombin and thrombin receptor in terms of hemostasis and hematopoiesis. The effects of prothrombin- and thrombin receptor-deficiencies on cell proliferation and differentiation will be studied directly in double knockout ES cells and knockout mice. If mice lacking prothrombin or the thrombin receptor are not viable, double knockout ES cells will be used to prepare chimeric mice to determine specifically which tissues can tolerate deficiencies in these proteins. Immunohistochemistry, together with in situ RNA hybridization, will be used to characterize the expressions of prothrombin and the thrombin receptor in normal or chimeric mice during development. Thrombin receptor deficient ES cell lines and tissues from deficient mice will be studied to determine whether additional protease activated receptors can be identified. These studies pursue a direct approach to define the biological functions of prothrombin and the thrombin receptor in growth, development and hemostasis.